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Abstract

BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important
serine/threonine-protein kinase. Two domains on BRaf can independently bind its
upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain
(RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers
to compare the Ras binding process in two pathological mutants of BRaf
responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two
mutants differ in their kinetics of Ras-binding, though both bind Ras with
similar increased overall affinity. BRaf (A246P) exhibits a slightly higher
Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force
versus the wild type. The contrary phenomenon is observed in the Q257R mutation.
Simulations of the unstressed-off rate, koff(0),
yield results in accordance with the changes revealed by the mean unbinding
force. Our approach can be applied to rapidly assess other mutated proteins to
deduce the effects of mutation on their kinetics compared to wild type proteins
and to each other.

Diagram of the experimental setup. The beam expander comprises L1 and
L2. L3 is a coupling mirror, which focuses the collimated laser beam
at the conjugate point of the tube mirror (L4) and the objective of
the microscope. The image of the bead is projected to the quadrant
photodiode detector (QD) by L5. An acousto-optic deflector (AOD) is
employed to manipulate the beam during calibration. The AOD is
controlled by a field programmable gate array (FPGA), which is
driven by a direct digital synthesizer (DDS).

(a) Image of beads acquired by CCD (left: a trapped Ras-bead; right:
a fixed BRaf-bead. Bead diameter: 5 μm). (b) Diagram of the optical
tweezers setup for measuring the unbinding force between trapped
Ras-beads and fixed BRaf-beads. The fixed BRaf-bead is brought in
contact with the trapped bead by the PZT. (c) If binding between the
beads occurs, the trapped bead deviates from the equilibrium
position as the fixed bead is pulled back. (d) When the gradient
force on the trapped bead is strong enough to break the association,
unbinding occurs.

A typical unbinding trace acquired by the QD for measurement (The
voltage output of the QD has been converted to force using Eq. (6).). I:
Corresponding to the process illustrated in Fig. 3(c). II: Corresponding to the process
illustrated in Fig. 3(d).

Histogram of unbinding force for Ras/BRaf and Ras-GTP/BRaf (A470)
interactions and the fitting curve of the experimental data. Insert:
Adhesion ratios for Ras-GTP/BRaf, Ras-GTP/BRaf (A246P), Ras-GTP/BRaf
(Q257R), Ras/BRaf and Ras-GTP/BRaf (A470) interactions. The adhesion
ratios of three specific interaction groups all exceeded 20%, while
those of control groups were all around 10%. “n” referred to the number
of contact cases measured in each group.

(a) Unbinding force histograms and simulated curves for the Ras-GTP/BRaf
interaction, which include 585 binding-unbinding events among 1800
attachment events. (b) Ras-GTP/BRaf (A246P) interactions, which include
596 binding-unbinding events among 2536 attachment events. (c)
Ras-GTP/BRaf (Q257R) interactions, which include751 binding-unbinding
events in 2269 attachment events. The arrow indicates the position of
each specific binding peak. The fitting of nonspecific interactions is
according to Eq. (7). The
fitting of binding peak is according to Eq. (11)-(13).